Odkazy
Abrin And Ricin - Two Dangerous Poisonous Proteins
Jiri Patocka
Department of Toxicology, Military Medical Academy,
Hradec Kralove, Czech Republic
E-mail: patocka@pmfhk.cz
Introduction
Abrin and ricin are natural toxic protein toxins
isolated from plant seeds [1], i.e., they are phytotoxins. Both proteins
are composed of two peptide chains, signified as A-chain and B-chain, which
are linked by a disulphide bond. Both toxins have large scale molecular
similarity, and the A-chains of abrin and ricin have a 102 conserved amino
acid homology; it is possible to create hybrid toxins (in a reciprocal
manner) between the A- and the B-chains of abrin and ricin. Also, the mechanism
of their toxic action is the same. The A-chain inhibits protein synthesis,
whereas the B-chain binds to cell surface receptors containing terminal
galactose and acts as an immunotoxin. The A-chain contains the toxic activity
and the B-chain gives the toxin a cell recognition and binding function
to facilitate transport across the cell membrane. The A-chain is not active
until it is internalized by the cell, where it halts protein synthesis.
The extreme toxicity of both abrin and ricin and their relative ease of production make them potential chemical warfare agents or terrorist weapon. Their production is easy and available on large scales from natural sources or by modern biotechnology processes, which may produce toxins that were previously difficult to obtain in significant quantities. Ricin is included in Schedule 1 of the Chemical Weapons Convention (CWC). Both toxins are included in the latest version of The Biological and Toxin Weapons Convention (BTWC) Procedural Report and Rolling Text: Ad Hoc Group 23rd session (23 April - 11 May 2001).
ABRIN
Abrin is a potent toxin that has been isolated from the seeds of Abrus
precatorius (or Rosary pea). Its use as a tool for research was described
in 1972 by Sharon and Lis [2]. Abrin exists in two forms, abrin a and abrin
b. Both are composed of two chains, an A-chain and a B-chain. A disulfide
bond between Cys247 of the A-chain and Cys8 of the B-chain links the A
and B chains. The A-chain is 251 residues and is divided into 3 folding
domains. The A-chain catalytically inactivates 60S ribosomal subunits by
removing adenine from positions 4 and 324 of 28S rRNA therefore inhibiting
protein synthesis. The B-chain is a galactose specific lectin that facilitates
the binding of abrin to cell membranes [3,4]. The B-chain of both forms
of abrin consist of 268 amino acid residues and share 256 identical residues
[5]. Comparison of their sequences with that of the ricin's B-chain shows
that 60% of the residues of abrin's B-chain are identical to those of the
ricin's B-chain and that two saccharide-binding sites in ricin B-chain
identified by a crystallographic study are highly conserved in abrin B-chain
[5].
The mechanism of toxic action of abrin is identical to that of ricin, but the toxicity of abrin in mice is 75 times that of ricin (0.04 µg/kg for abrin compared to3 µg/kg for ricin.) The diagnosis, clinical features, treatment, protection, prophylaxis and so on is also the same for both abrin and ricin intoxications [6].
RICIN
Ricin was found by Stillmark in 1889 [7] as the first plant lectin from the seeds of the castor plant, Ricinus communis. As with abrin, ricin is a lectin consisting of two polypeptide chains, the A-chain (30 kDa) and the B-chain (32 kDa), linked by a disulfide bond. It is one of a group of dichain ribosome-inactivating proteins, which are specific for the depurination of a single adenosine in ribosomal ribonucleic acid [8] The A-chain of ricin has the ability to modify catalytically the 28S subunit of ribosomes to block protein synthesis. The lectin subunit, B chain, of ricin plays an important role of binding to the cell surface glycoconjugates of target cells and facilitates the internalization and translocation of the toxin to cytosol [9]. The structure of ricin is given in Figure 1, taken from reference 9. The A-chain is the on the upper right and the B-chain on the lower left.
Toxicity
The toxicity of castor beans has been known since ancient times, and more than 750 cases of intoxication in humans have been described [10]. There is a 100-fold variation in the lethal toxicity of ricin for various domestic and laboratory animals, per kilogram of body weight. Of animals tested, the chicken and frog are least sensitive, while the horse is the most sensitive[11]. Toxicity of ricin also varies with route of challenge. In laboratory mice, the approximate dose that is lethal to 50% of the exposed population (LD50) and time to death are, respectively, 3 to 5 µg/kg and 60 hours by inhalation, 5 µg/kg and 90 hours by intravenous injection, 22 µg/kg and 100 hours by intraperitoneal injection, 24 µg/kg and 100 hours by subcutaneous injection, and 20 mg/kg and 85 hours by peroral administration. Low oral toxicity reflects poor absorption of the toxin from the gastrointestinal tract.
Mechanism of Toxicity
Ricin is very toxic to cells. Both ricin and abrin inhibit protein synthesis by inactivating the ribosomes. The B-chain binds to cell surface receptors and the toxin-receptor complex is taken into the cell; the A-chain has endonuclease activity and extremely low concentrations will inhibit protein synthesis. Figure 2 is a schematic showing the mechanism of toxic action for ricin.
Signs and Symptoms
The clinical picture, clinical signs, symptoms, and pathological manifestations in intoxicated victims depends on the route of exposure [12]. When inhaled as a small particle aerosol, ricin produces symptoms within 8 hours. Respiratory distress, fever, cough, dyspnea, nausea, and chest tightness are followed by profuse sweating, the development of pulmonary edema, cyanosis, hypotension, and finally respiratory failure and circulatory collapse. Time to death ranges from 36-72 hours, depending on the dose received. Ingestion of ricin causes gastrointestinal signs and gastrointestinal hemorrhage with necrosis of liver, spleen, and kidneys; intramuscular intoxication causes severe localized pain, muscle and regional lymph node necrosis, and moderate involvement of visceral organs. Transient leukocytosis appears to be a constant feature in humans, whether intoxication is via injection or oral ingestion. Leukocyte counts are 2- to 5-fold higher than the normal value. Children are more sensitive than adults to fluid loss, due to vomiting and diarrhea, and can quickly become severely dehydrated and die. If death has not occurred in 3-5 days, the victim usually recovers.
Diagnosis
Like other potential intoxications on the unconventional battlefield, epidemiological findings will likely play a central role in diagnosis. The observation of multiple cases of very severe pulmonary distress in a population of previously healthy young soldiers, linked with a history of their having been at the same place and time during climatic conditions suitable for biological warfare attack, would be suggestive of ricin intoxication. The differential diagnoses of aerosol exposure to ricin would include staphylococcal enterotoxin B, exposure to pyrolysis by-products of organofluorine polymers [13] or other organohalides, oxides of nitrogen, and phosgene. Laboratory findings are nonspecific but similar to other pulmonary irritants that cause pulmonary edema. Enzyme-linked immunosorbent assays in blood or other body fluids [14] or immunohistochemical techniques may be useful in confirming ricin intoxication but identification in body fluids or tissues is difficult.
Medical Management
Management of ricin-intoxicated patients depends on the route of exposure. Patients with pulmonary intoxication are managed by appropriate treatment for pulmonary edema and respiratory support. Gastrointestinal intoxication is best managed by vigorous gastric decontamination with activated charcoal, followed by use of cathartics such as magnesium citrate. Volume replacement of gastrointestinal fluid losses is important. In percutaneous exposures, treatment would be primarily supportive.
Immunization and Passive Protection
Animal studies have shown that either active immunization or passive prophylaxis or therapy is extremely effective against intravenous or intraperitoneal intoxication with ricin. On the other hand, inhalational exposure is best countered with active immunization or prophylactic administration of aerosolized specific anti-ricin antibody [15]. Unfortunately, effective countermeasures for human use are not available at this time.
Supportive and Specific Chemotherapy
No specific treatment exists, and care is thus supportive. In cases of gastrointestinal exposure, gut decontamination via lavage, activated charcoal, and cathartics is recommended. For inhalant intoxication, supportive therapy to counteract acute pulmonary edema and respiratory distress is indicated. Symptomatic care is the only intervention presently available to clinicians for the treatment of incapacitating or lethal doses of inhaled ricin. Large amounts of volume replacement may be necessary. Intensive respiratory therapy, fluid and electrolyte replacement, anti-inflammatory agents, and analgesics would likely be of benefit in treating aerosol-intoxicated humans.
Protection and Prophylaxis
A protective mask offers protection from aerosol exposure. Because of the extreme toxicity of these toxins, a relatively small leak could easily result in a significant exposure. Eyes should be protected when possible. Definitive studies have not been done to assess the effects of aerosolized toxins on the eyes. Donning the protective mask prior to exposure would, of course, protect the eyes. Because important threat biological/toxin warfare agents are not dermal active, special protective clothing, other than the mask, is less important in at toxin attack than a chemical attack. Presently available clothing should be effective against biological threat as we know them. Although a vaccine is not currently available, candidate vaccines are under development that are immunogenic and confer protection against lethal aerosol exposures in animals. Prophylaxis with such a vaccine is the most promising defense against a biological/toxin warfare attack with ricin.
Decontamination
Ricin may be inactivated with 0.5% hypochlorite.
Since it is not dermal active and is involatile, decontamination may not
be as critical as with certain other biological and chemical agents.
ABRIN AND RICIN AS CHEMICAL WARFARE
OR TERRORIST AGENTS
Ricin is the only toxin to exist naturally in
large quantities. It is a byproduct of castor oil production and ricin
isolation is a simple and cheap separation. Easy preparation and low price
might make this toxin attractive to poor country. Abrin may be considered
to be an available toxin for weaponizing because its source, Abrus precatorius,
may be easily cultivated and the preparation of the pure toxin is not complicated.
For nations or terrorists who lack the money to spend on nuclear weapons
and other high-tech killing instruments, toxin warfare offers horrific
appeal: biological/toxin weapons are cheap, easy to make, and simple to
conceal. Even small amounts, if effectively used, could cause massive injuries
and make many suffer [16].
Note: Ricin’s significance as a potential biological warfare toxin relates in part to its wide availability. Worldwide, one million tons of castor beans are processed annually in the production of castor oil and in the waste is five percent ricin by weight. The toxin is also quite stable and extremely toxic by several routes of exposure, including the respiratory route. Ricin is said to have been used in the assassination of Bulgarian exile Georgi Markov in London in 1978. Markov was attacked with a specially engineered weapon disguised as an umbrella which implanted a ricin-containing pellet into his body. [ASA 93-1]
POSSIBILITIES OF REAL-TIME DETECTION OF A TOXIN ATTACK
Real-time detection of toxins is very important,
because without timely warning, the most effective generic countermeasure
of soldiers, the protective mask, may be of limited value. There have been
successful efforts in the past to develop real-time detectors of a chemical
agent attack. It will be more difficult to develop such detectors for toxins
for several reasons. As stated above, toxins must be presented as inspirable
aerosols, which act as a cloud, not as vapors or droplets, as the chemical
agents. Toxin detectors working at the present state of technology would
likely have to have the specificity of immunoassays to identify a toxin
and differentiate it from other organic material in the air. Continuous
monitoring by such equipment would be extremely costly and logistically
very controversial. Analytical assays would necessarily be more complex
and less likely to identify distinct toxins, but might detect that something
unusual was present. This might be almost possible on a battlefield, but
would be nearly impossible in the case of a terrorist attack. We must hope
that future advances in technology could well resolve our present technical
difficulties.
